Exposure to cobalt may occur through diet, dietary supplements, occupational exposure, biomedical applications and medical devices [10]. Although not completely declared some dietary supplements are promoted as performance-boosting nutrients and contain soluble and bioavailable Co as their active ingredient [11]. As cobalt supplements may induce erythropoietin production thus affecting endurance performance, in 2017 the World Anti-Doping Agency included them in its prohibited list [12]. The erythropoietic effect of cobalt in our study was demonstrated by elevated Hb content and increased RBCs in almost all experimental groups. The expanded erythropoiesis may be due increased erythroferrone production as it is stimulated by hypoxia [13]. In addition, stimulated RBC production may be also due upregulation of GATA-1 known to enhance erythroid differentiation and to be highly expressed under hypoxia [14, 15]. Although the dose of CoCl2 sufficient to stimulate erythropoiesis is unknown, Hoffmeister et al. show that oral Co2+ dosage of 10 mg/day for 5-day duration exerts erythropoietic effects [16]. In their earlier work the same authors find that a 3-week administration of 5 mg/day Co supplement significantly increased hemoglobin mass while the other hematological parameters were not significantly affected [17].
In serum Co2+ binds to albumin, and the concentration of free, ionized Co2+ is estimated at 5–12% of the total cobalt concentration. Our results for chronic treatment with CoCl2 suggest that immature mice accumulate ~ 4.4-fold more metal compared to mature animals. Also, during long-term exposure the most Co is accumulated in serum after 30 days of exposure and decreases by day 90 of dosing. The decrease may be due to “auto-inhibition” by cobalt as suggested by Simonsen et al. [18], and increased excretion and/or suppressed absorption due to elevated body iron. Our results are in accordance with those of Reuber et al. [19] who demonstrate enhanced Co excretion during Co and/or iron supplementation. Our experimental data suggest that serum Co concentration is a reliable marker for a recent exposure - up to 30 days.
Research data imply that during long-term cobalt exposure in vivo cobalt will be taken up practically irreversibly in the red cells during their 120 days life span [20]. Our results demonstrate significant cobalt accumulation in RBCs following chronic exposure to CoCl2. The highest concentration was found in day 30 mice and decreased by day 90 of exposure. Our results for the highest Co concentration in RBCs of day 30 mice are also similar to those of Bryan et al. [21] where cobalt concentration in erythrocytes increased with time to reach a plateau after 5–6 weeks of daily peritoneal exposure of rats.
At systemic and cellular level Co is sequestered along the Fe-acquisition pathway. The behavior of the transferrin-Co complex is similar to that of iron with a fast interaction with the C-lobe and a very slow one with the N-lobe of TfR1 [22].
Iron is biologically essential but toxic in high concentrations. For this reason, its metabolism is tightly controlled at cellular and systemic level to prevent deficiency or overload [5].
In our study, chronic in vivo exposure to CoCl2 significantly altered serum and erythrocyte Fe content. Similar results for significantly increased Co and Fe concentrations in the serum and RBCs of Co-exposed mice were obtained in our previous studies using lower daily dose of CoCl2 [23].
Although Fe significantly increased in serum of Co-exposed mice, its content in mature mice remained lower compared to immature, with the largest difference of 9.9% between d30 and d90 mice. A possible explanation for the lower serum Fe content in mature mice is the elevated Hb content suggesting Fe utilization in heme synthesis. RBC iron decreased in Co-exposed mature mice but increased in d30 mice, being ~ 2.48-fold higher compared to d90 mice.
Our results for reduced erythrocyte Fe content indicate that erythroid cells possibly export Fe to survive. This hypothesis is supported by Keel et al. [24] who demonstrate that the export of excess cytoplasmic heme from the erythroid precursors is mediated by the feline leukemia virus, subgroup C, receptor (FLVCR). Iron overload is known to damage RBC plasma membranes by increasing reactive oxygen species production and osmotic fragility [25]. A possible mechanism for erythrocyte Fe release in the blood plasma may be through increased ferroportin activity. Zhang et al. [25] demonstrate abundant ferroportin expression in mature RBCs and hypothesize that erythrocytes export Fe to avoid iron overload and hemolysis.
In our studies we have observed stimulated ferroportin expression in the bone marrow of day 60 and day 90-cobalt treated mice (our unpublished data). In addition, cellular iron export is stimulated by ceruloplasmin. Low oxygen concentrations stimulate Fe release by ceruloplasmin who is also known to stabilize ferroportin on cell membrane [26]. Mature erythrocytes express ceruloplasmin receptors [27] and therefore its role in Fe reduction by RBCs should not be excluded. The insignificant change in cell volume also suggests that the erythrocytes likely export the excess Fe. According to McLaren et al. [28], cases of excess Fe are associated with RBC’s expanded size and higher cellular Hb concentration as a functional utilization of Fe within a non-toxic compartment.
Within the cells Fe is stored in the form of ferritin. Ferritin is an iron-binding protein that indicates total body iron stores. The iron-storing capacity of ferritin is assisted by the ferroxidase activity of ferritin heavy chain, which converts reactive iron (Fe2+) into inert, nucleated iron (Fe3+), no longer available to catalyze the production of free radicals via Fenton chemistry [29]. It is an acute-phase protein that increases in response to inflammatory states, including malignancy, infection, and in liver, renal and autoimmune diseases. Serum ferritin is a clinical marker for Fe status as it is increased in cases of Fe overload and reduced in Fe deficiency [30]. Elevated serum ferritin also reflects macrophage ferritin content as ferritin is predominantly secreted in the circulation by the macrophages [5]. In our study serum ferritin was elevated almost in all experimental groups following chronic exposure to CoCl2. In day 90 exposed mice serum ferritin concentration was increased more than 5-fold also suggesting enhanced loss of Fe by the erythrocytes.
Another key marker for Fe trafficking is TfR1. It can bind two different Fe-binding molecules: transferrin and ferritin [3]. Serum contains a soluble TfR (sTfR) molecule that circulates bound to transferrin that is cleaved from the whole TfR molecule and is a sensitive marker for erythropoiesis and iron deficiency [31]. The circulating sTfR level reflects total body TfR concentration, the major source of sTfR being bone marrow erythroid precursors [3].
The reduced serum TfR1 concentration in our study is in accordance with the increased serum Fe and ferritin concentrations. These results suggest downregulation of TfR due to increased intracellular Fe storage and total body Fe as TfR expression is shown to decrease in cases of excess Fe [3]. According to R’zik and Beguin [32] there is an inverse correlation between body iron stores and total TfR. In addition, the high serum Fe concentration may counteract erythropoietin production by the kidneys resulting in lower TfR expression.
Another explanation for the reduced TfR1 may be that the increased serum ferritin delivers Fe to the tissues serving as an alternative pathway to the Tf-TfR one, as observed by Wang et al. [30]. Also, in vitro experiments have shown that the isoform of the second transferrin receptor - TfR2-α, incorporates Tf-bound iron into cells as effectively as TfR1, and it is expressed predominantly by erythroid progenitors but the expression of TfR2 mRNA does not show obvious changes upon iron loading or iron chelation [3]. In our previous study we also demonstrate stimulated TfR2 tissue expression in immature mice following Co exposure [33]. This suggests that the role of TfR2 should not be neglected in cases of stimulated erythropoiesis and/or elevated serum Fe.
Increased serum iron levels upregulate hepcidin expression. Hepcidin acts as a negative regulator of Fe. In coordination with ferroportin hepcidin regulates Fe entry into plasma, its utilization and storage [6]. The elevated serum Fe in our study enhanced hepcidin production in almost all Co-exposed groups. The stimulated erythropoiesis in day 18 and day 60 mice may be a possible explanation for the significantly reduced serum hepcidin in these groups. In addition, erythroferrone which stimulated RBC production suppresses hepcidin expression in a time-dependent manner [13]. Stimulated erythropoiesis strongly suppresses the production of hepatic hepcidin in mice and humans, allowing more dietary iron and Fe from stores to enter blood plasma for heme and hemoglobin synthesis by developing erythrocytes in the marrow [5; 7].
The results indicate that Fe metabolism is tightly controlled/regulated by erythropoiesis through the suppression of hepcidin. In our study serum hepcidin concentration in mature mice was lower than in the immature. Possible explanation for this may be that during chronic exposure the high extracellular Fe concentration may suppress hepcidin production by a feedback mechanism.